The culturable bacterial population and phospholipid fatty acid (PLFA) profile of casing soil were investigated at different mushroom (Agaricus bisporus) cropping stages. The change in soil bacterial PLFAs was always accompanied by a change in the soil culturable bacterial population in the first flush. Comparatively higher culturable bacterial population and bacterial PLFAs were found in the casing soil at the primordia formation stage of the first flush. There was a significant increase in the ratio of fungal to bacterial PLFAs during mushroom growth. Multivariate analysis of PLFA data demonstrated that the mushroom cropping stage could considerably affect the microbial community structure of the casing soil. The bacterial population increased significantly from casing soil application to the primordia formation stage of the first flush. Casing soil application resulted in an increase in the ratio of gram-negative bacterial PLFAs to gram-positive bacterial PLFAs, suggesting that some gram-negative bacteria might play an important role in mushroom sporophore initiation. The culturable bacterial population and phospholipid fatty acid (PLFA) profile of casing soil were investigated at different mushroom (Agaricus bisporus) cropping stages. The change in soil bacterial PLFAs was always accompanied by a change in the soil culturable bacterial population in the first flush. Comparatively higher culturable bacterial population and bacterial PLFAs were found in the casing soil at the primordia formation stage of the first flush. There was a significant increase in the ratio of fungal to bacterial PLFAs during mushroom growth. Multivariate analysis of PLFA cropping stage could significantly affect the microbial community structure of the casing soil. The bacterial population increased significantly from casing soil application to the primordia formation stage of the first flush. Casing soil application resulted in an increase in the ratio of gram-negative bacterial PLFAs to gram-positive bacterial PLFAs, suggesting that some gram-negative bacteria might play an important role in mushroom sporophore initiation.